Product formation kinetics in cell culture

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Categorization of product formation

Product formation

 Directly coupled with energy metabolism Growth associated product

 Indirectly coupled with energy metabolism Mixed-growth-associated produ

 Not coupled with energy metabolism

Non-growth associated produc

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Classification of Low-Molecular-Weight Fermentation Products

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General rate expression for product formation

In general, the rate of product formation, irrespective of the class of product is given by

rP  volumetric rate of product formation kg m-3 s-1,

𝑟𝑃 = 𝑞𝑃 𝑥 x  biomass concentration, and
qP specific rate of product formation with dimensions T-1.

qP can be measured by measuring the rate of product formation and biomass concentration

1 𝑑𝑃 1 𝑑𝑥 𝑑𝑃
𝑞𝑃 = = = 𝜇𝑌𝑃/𝑋
𝑥 𝑑𝑡 𝑥 𝑑𝑡 𝑑𝑥

qP not necessarily constant during batch culture

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a) Growth associated product

 The rate of production related to cellular energy demand for products formed using
pathways that generate ATP

 If production is coupled to energy metabolism,

product will be formed whenever there is growth

 ATP also required for other activities called maintenance

such as cell motility, adjustment of membrane potential
and internal pH

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 Carry out of maintenance functions  ATP requirement  product formation

Therefore, kinetic expression for growth associated product formation (rP) must include
maintenance associated product formation in addition to growth associated production

𝑟𝑃 = 𝑌𝑃𝑋 𝑟𝑋 + 𝑚𝑃 𝑥 rX  volumetric rate of biomass formation,

YPX theoretical or true yield of product from biomass,
mP  specific rate of product formation due to
𝑟𝑃 = (𝑌𝑃𝑋 µ + 𝑚𝑃 )𝑥 ;Since 𝑟𝑋 = µ 𝑥
maintenance,

𝑞𝑃 = 𝑌𝑃𝑋 µ + 𝑚𝑃 ;Since 𝑟𝑃 =𝑞𝑃 𝑥 x  biomass concentration

mP has dimensions T-1

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b) Nongrowth-associated product

Produced in stationary phase when specific growth rate µ = 0

Thus specific product formation rate

qP = constant

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c) Mixed growth associated product

Product formation takes place during the slow growth and stationary phases.

Luedeking–Piret equation

𝑞𝑃 = 𝛼𝜇𝑔 + 𝛽

if
α = 0  non-growth associated product
β = 0  growth associated product; then α = 𝑌𝑃𝑋

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Kinetics of substrate uptake in cell culture

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 Apparent growth yield

𝑥 − 𝑥0
𝑌𝑋/𝑆 =
𝑠0 − 𝑠
Where x, x0, s and s0 are measured biomass concentrations

YX/S is typically 0.4 to 0.6 g/g for most yeast and bacteria

Maintenance coefficient mS is used to describe specific substrate uptake rate for cellular maintenance
1 𝑑𝑠
𝑚𝑆 = ( )𝑚
𝑥 𝑑𝑡
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General rate expression for substrate consumption

𝑑𝑠
𝑟𝑆 = 𝑞𝑆 𝑥 =
𝑑𝑡

𝑑𝑆 1 𝑑𝑥
=
𝑑𝑡 𝑌𝑋/𝑆 𝑑𝑡

where
rS volumetric rate of substrate consumption with units of, for example, kg m-3 s-1,
qS the specific rate of substrate uptake, (s-1 or h-1) and
x biomass concentration. Like

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Substrate Uptake in the Absence of Extracellular Product Formation

With the assumption that all the substrate is used for growth and maintenance activities

𝜇
𝑟𝑆 = 𝑥 + 𝑚𝑆 𝑥
𝑌𝑋/𝑆

𝜇𝑚 𝑆 𝑥
𝑟𝑆 = + 𝑚𝑠 𝑥
𝑌𝑋/𝑆 (𝐾𝑆 + 𝑆)

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 Substrate consumption with extracellular product formation

Product formation directly linked Product formation not directly linked

to energy metabolism to energy metabolism

No separate term for substrate consumption Separate term for substrate consumption
in the Rate expression in the Rate expression

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Product synthesis indirectly coupled to energy metabolism

𝑟𝑥 𝑟𝑃
𝑟𝑆 = + + 𝑚𝑆 𝑥
𝑌𝑋/𝑆 𝑌𝑃/𝑆
rS volumetric rate of substrate consumption,
rX  volumetric rate of biomass production,
rP  volumetric rate of product formation,
YXS  true yield of biomass from substrate,
YPS  true yield of product from substrate,
mS  maintenance coefficient, and
x is the biomass concentration

𝜇 𝑞𝑃
𝑟𝑆 = + + 𝑚𝑆 x
𝑌𝑋/𝑆 𝑌𝑃/𝑆

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Effect of maintenance on yields

 True /theoretical (YX/S, YP/S and YPX) are difficult to evaluate

 True yields are essentially stoichiometric coefficients, which is known only
for simple fermentations.
 But theoretical yields can be related to observed yields

Observed yields

−𝑑𝑋 𝑟𝑋 −𝑑𝑃 𝑟𝑃 −𝑑𝑃 𝑟𝑃

𝑌′𝑋/𝑆 = = 𝑌′𝑃/𝑋 = = 𝑌′𝑃/𝑆 = =
𝑑𝑆 𝑟𝑆 𝑑𝑋 𝑟𝑋 𝑑𝑆 𝑟𝑆

where X, S, and P masses of cells, substrate, and product, respectively, and

rX, rS, and rP observed rates evaluated from experimental data.
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Observed biomass yield from substrate

𝒓𝒙 𝝁𝒙
𝒀′𝑿/𝑺 = = Assuming that no extracellular product formation/
𝒓𝑺 𝝁 Product directly coupled to energy metabolism
+ 𝒎𝑺 𝒙
𝒀𝑿/𝑺

Where YX/S theoretical yield and Y’X/S apparent/observed yield

Taking reciprocal of the above expression

𝟏 𝟏 𝒎𝑺
= +
𝒀′𝑿/𝑺 𝒀𝑿/𝑺 𝝁

 The above expression is generally not applicable to batch growth data

 Observed yield can be improved by decrease in maintenance coefficient

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Observed product yield from biomass

𝒓𝑷 𝝁𝒀𝑷𝑿 + 𝒎𝑷 𝒙 Assuming that product formation not directly coupled

𝒀′𝑷/𝑿 = =
𝒓𝒙 𝝁𝒙 to energy metabolism

Where YP/X theoretical yield and Y’P/X apparent/observed yield

𝒎𝑷
𝒀′𝑷/𝑿 = 𝒀𝑷/𝑿 +
𝝁

Observed yield can be improved by increasing the mP and decreasing µ

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Observed product yield from substrate

𝒓𝑷 𝝁𝒀𝑷/𝑿 +𝒎𝑷 𝒙 𝝁𝒀𝑷/𝑿 +𝒎𝑷

𝒀′𝑷/𝑺 = = 𝝁 = 𝝁
𝒓𝑺 +𝒎𝑺 𝒙 +𝒎𝑺
𝒀𝑿/𝑺 𝒀𝑿/𝑺

Y’P/S can be improved by decreasing the specific growth rate µ or by increasing mP

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